The present invention relates to electrodes for detecting nucleic acid hybridization and to the method of detecting nucleic acids utilizing such electrodes. The electrodes may also be used for detection of proteins.
The detection of nucleic acid hybridization at solid surfaces has been used for the identification of infectious organisms in clinical specimens (Spargo, C. A. et al., (1993), Molecular and Cellular Probes 7, 395-404; Martin, W. J. (1994) Infectious Diseases. In The Polymerase Chain Reaction (K. B. Mullis, F. Ferre and R. A. Gibbs, eds.), pp. 406-417. Berkhauser, Boston), the quantitation of mRNA for gene expression analysis (Schena, M., et al., (1995). Science 270, 467-470), and the sequencing or resequencing of genomic DNA on high-density "chip" arrays (Chee, M., et al., (1996) Arrays. Science 274, 610-613). Presently, these efforts involve the attachment of a fluorescent label to the target nucleic acid, which is then hybridized with a probe-modified surface and detected after washing the unhybridized DNA away from the solid surface. Since detection of photons is required to signal hybridization, analysis of high-density arrays labeled in this manner requires high-resolution fluorescence microscopes. Alternatively, indirect detection of hybridization can be accomplished using sandwich assays where the surface-bound hybrid is subsequently hybridized to an additional signal probe that carries one or more fluorescent labels or enzymes that convert a non-fluorescent substrate to a fluorescent one (Spargo, C. A. et al., (1993), Molecular and Cellular Probes 7, 395-404). By attaching multiple enzymes to the signal probes, large signal amplification can be achieved (Holodniy, M. et al., (1995). J. Virology 69, 3510-3516); however, the preparation of these multiple enzyme systems is complex.
The patents of Heller (U.S. Pat. Nos. 5,532,129; 5,565,322; 5,605,662; and 5,632,957) disclose the use of an electrode with a permeation layer which is an agarose gel placed on the electrode. A potential is applied to the electrode that brings probe or target DNA to the reaction site on the electrode. Because of the high voltages required, there is a strong possibility of electrooxidation of the probe, target and other components of the solution. The gel protects the system so that excessive levels of target DNA do not accumulate and so that the DNA oxidation is minimized.
Labeled proteins and soluble mediators have been used to detect protein-protein interactions. For example, the patent of Weetall (U.S. Pat. No. 5,066,372) discloses a support layer on a working electrode that is porous to mediator and to which protein can be immobilized. See also U.S. Pat. Nos. 4,945,045 of Hill, 4,545,382 of Higgins, and 5,378,628 of Gratzel. The instant invention differs from these references in that it utilizes guanine as a electron donor to drive the electron transfer reaction rather than utilizing an enzyme oxidation reaction.
The paper of Wang et al. (Wang et al., (1997), Anal. Chem. 69, 4056-4059), describes a membrane-covered carbon electrode for analysis of oligonucleotides in the presence of polymeric nucleic acids. The purpose of the membrane is to exclude the polymeric DNA, while small molecules can pass through the membrane for electroanalysis by the carbon electrode. The membrane is not used for attachment of probes and the membrane-covered electrodes do not offer discrimination at the sequence level.
The parent applications, whose entire specifications, drawings, and claims are specifically incorporated herein by reference, disclose, among other inventions, sequencing and methods of qualitatively and quantitatively detecting nucleic acid hybridization.
Such inventions represent a major advance in the art and provide oxidation-reduction complexes which function in a catalytic manner without the addition of an enzyme or fluorescent label, provide for a catalytic current to give the concentration of guanine, or alternate base, in a manner useful for determining the presence or absence of a target, and provide for extremely accurate testing.
Although a major advance, the prior inventions rely on electrodes that are not entirely suitable for measuring the oxidation-reduction reaction. The invention herein provides electrodes coated with a polymer material (a) to which oligonucleotide probes can be covalently attached; (b) in which the soluble mediator can diffuse freely and dock with immobilized DNA; (c) for which no additional electrochemical current is generated at potentials between 0 and +1.3 V (vs a Ag/AgCl reference electrode); and (d) at which the immobilized oligonucleotide probe is available to capture target nucleic acid. In addition, the polymer coating should preferably not adsorb protein or other non-targeted nucleic acid material in biological samples, unlike previously developed electrodes that utilize nylon or nitrocellulose films that have significant affinity for protein and non-complementary nucleic acid. Adsorbed protein, non-targeted nucleic acid, and other biological material contribute to the undesirable background signal and may inhibit proper capture of targeted nucleic acid.
The distinguishing feature of the approach of the invention is that the unlabeled target DNA is differentiated from the synthetic probes by modulation of the electron-transfer reactivity. In the use of the polymer-electrode of the invention, instead of differentiating duplex and single-stranded DNA (as in ethidium bromide fluorescence (Waring, M. J., (1965). J. Mol. Biol. 13, 269), we differentiate probe and target. The importance of this difference is that our assay is not dependent on bringing double-stranded DNA to the electrode. Thus, the invention is equally well suited for detection of single- or double-stranded DNA. More importantly, single-stranded RNA, which is of particular interest in identifying unamplified genomic RNA from viruses such as HIV or Hepatitis C, in detecting ribosomal RNA from bacteria, or in quantitating cellular MRNA for gene expression analysis, may also be detected. For the studies described here, the probes are attached to the membrane via the endogenous exocyclic amines of the nucleobases, as has also been done for direct attachment to glassy carbon electrodes (Millan, K. M. et al., (1994), Anal. Chem. 66, 2943-2948; Millan, K. M. & Mikkelsen, S. R. (1993). Anal. Chem. 65, 2317-2323). Membranes behave similarly with synthetic oligonucleotides to which an alkyl amine linker is appended, which can provide for greater hybridization efficiency and specificity.
Other objects and advantages will be more fully apparent from the following disclosure and appended claims.